Dirofilaria immitis and Dirofilaria repens in mosquitoes from Corsica Island, France

Background Dirofilaria immitis and Dirofilaria repens are the main causative agents of heartworm disease and subcutaneous dirofilariasis in domestic and wild canids, respectively. Both pathogens have zoonotic potential and are transmitted by mosquitoes. The present study aimed to determine the transmission period, prevalence and diversity of Dirofilaria spp. vectors from endemic areas of Corsica (France). Methods A monthly point data model based on average temperature recorded by four meteorological stations during 2017 was used to calculate the Dirofilaria transmission period. From June to September 2017, female mosquitoes (n = 1802) were captured using Biogents® Sentinel 2 traps lured with carbon dioxide and BG-Lure™ or octanol. Mosquitoes were identified to species level, pooled accordingly, and screened using multiplex real-time qPCR to detect D. immitis and D. repens. Results The monthly point data model showed the possible transmission of Dirofilaria spp. from the third week in May to the last week in October in the studied area. Mosquitoes were identified as Ochlerotatus caspius (n = 1432), Aedes albopictus (n = 199), Culex pipiens sensu lato (n = 165) and Aedes vexans (n = 6) and were grouped into 109 pools (from 1 to 27 specimens, mean 11.4 ± 0.7), of which 16 scored positive for Dirofilaria spp. (i.e., n = 13; estimated infection rate [EIR] = 1.1% for D. immitis and n = 3; EIR = 0.2% for D. repens). Specifically, 6 (i.e., EIR = 3.8%) of 15 pools of Ae. albopictus were positive for D. immitis, 2 of 14 of Cx. pipiens s.l. were positive for D. immitis and D. repens, respectively, and 8 of 77 pools of Oc. caspius were positive for D. immitis (i.e., n = 6; EIR = 0.4%) and D. repens (i.e., 2; EIR = 0.1%). The highest mosquito infection rate was recorded in July (EIR = 2.5%), then in June (EIR = 1.3%) and September (EIR = 0.6%). Conclusions The data suggest that both Dirofilaria species are endemic and occur possibly in sympatry in the studied area in Corsica, highlighting the need to implement preventive chemoprophylaxis and vector control strategies to reduce the risk of these filarioids in dog and human populations. Graphical Abstract


Background
Dirofilaria immitis and Dirofilaria repens are zoonotic filarioid nematodes responsible for canine cardiopulmonary and subcutaneous dirofilariasis, respectively [1]. Dirofilaria immitis is of great veterinary importance while D. repens is the main causative agent of human dirofilariasis in the old world [2]. These mosquito-borne filarioids share the same definitive hosts (mostly canids), and several mosquitoes species (i.e., mosquitoes of the genera Culex, Aedes, Ochlerotatus, Anopheles, Coquillettidia, Armigeres, Mansonia and Psorophora) have been reported as competent vectors [3]. Several of these vectors feed indiscriminately on dogs and humans, resulting in a zoonotic sympatric occurrence in endemic areas [2].
Canine dirofilariasis is endemic in Southern Europe [23][24][25][26]. Corsica, a French island in the Mediterranean basin, is known as an epidemiological hotspot from which several cases of vector-borne diseases in humans and dogs are imported to the mainland. This is the case of D. immitis in dogs from Corsica [27,28] and human dirofilariasis caused by D. repens in visitors to the island [29]. Previous epidemiological studies confirmed the circulation of Dirofilaria spp. in human, dog and mosquito (i.e., Ae. albopictus) populations from the island [6,30,31]. However, data on the seasonality of transmission, diversity of the Dirofilaria vector and prevalence are sparse. This work advances understanding of the transmission period, prevalence and potential vectors of Dirofilaria spp. from endemic areas of Corsica (France).
One hundred and nine pooled (from 1 to 27, mean 11.4 ± 0.7) female specimens were prepared. For each mosquito pool, a 10-min bead-based mechanical lysis was performed in the TissueLyser apparatus in the presence of 800 µl of MEM medium (Sigma Aldrich). Mosquito lysates were centrifuged at 13,000g for 3 min, and genomic DNA was extracted from 200 µl of the supernatant in the presence of 100 µl of lysis buffer. Extraction was performed using the QIAcube kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. DNA was eluted in a final volume of 100 µl and stored at −20 °C until analysis. Finally, genomic DNA was analysed for the presence of Dirofilaria spp. using a multiplex real-time qPCR assay as described elsewhere [36].

Data analysis
Differences in Dirofilaria spp. infection were evaluated between mosquito species, sampling period and province using the analysis of covariance (ANCOVA) model within XLSTAT software (Addinsoft, Paris, France, 2018). The minimum infection rate (MIR) [37] and the estimated infection rate (EIR) [38] were calculated using the following formulas: MIR = (x/n)*100 and ERI = (1 − (1 − x/m) 1/k )*100, where x is the number of positive pools, n is the total number of mosquitoes tested, m is the number of mosquito pools and k is the average number of mosquitoes in each pool.

Results
The monthly point data model indicated the potential transmission of Dirofilaria spp. for 22 weeks (from the third week in May to the last week in October) in the study area (Fig. 1). During the summer season (from July to September), up to 2.5 mDG were recorded each month with a maximum activity of 3 mDG in August (Fig. 1). Morphological identification of mosquitoes revealed the presence of at least four species, dominated by Ochlerotatus caspius (n = 1432; 79.8%) followed by Aedes albopictus (n = 199, 11.0%), Culex pipiens sensu lato (n = 165; 9.2%) and Aedes vexans (n = 6; 0.3%). Most mosquitoes were caught in Solaro province (n = 1606; 89.1%), where the mosquito population was dominated by Oc. caspius (n = 1392; 86.7%) and Ae. albopictus (n = 168, 10.5%). Mosquito abundance in the other provinces ranged from 25 specimens in Borgo to 83 and 88 specimens in Aleria and Solenzara, respectively. The highest number of mosquitoes (n = 1496; 81.5%) was caught during September (Fig. 2).

Discussion
This study reports data on Dirofilaria spp. in mosquitoes collected in Corsica along with a prediction model to forecast the seasonal transmission of these filarioids, providing information about vector diversity and infestation rates with Dirofilaria spp. in Corsican mosquito species.
Dirofilaria transmission is related to an episystem complex involving several factors including temperature, vector and host abundance [2]. Data herein indicate that Dirofilaria transmission may occur over 22 weeks (from May to October), with maximum activity of 2.6 to 3 mDG during the summer period (June to August), as reported in the southern regions of Europe [1], especially in Italy [39]. Interestingly, in this study, the highest MIR/ERI were recorded from Ae. albopictus in July, which coincides with the first peak of seasonal transmission (more than 2.6 mDG). Moreover, during the highest transmission peaks (June and July), mosquito fauna was dominated by Ae. albopictus and Cx. pipiens s.l., which are well-known vectors for Dirofilaria spp., therefore representing an epidemiological risk for infection to dogs and humans. It is worth noting that those are the months when a large number of tourists visit the island, often along with their pets. The relationship between prediction and actual prevalence of Dirofilaria has already been confirmed in several studies [32,40,41]. However, this period could be extended by the presence of heat islands, microenvironments such as buildings and parking lots retaining heat during the day. Consequently, the extrinsic development of Dirofilaria larvae becomes possible during the cold season [2,42,43]. Furthermore, some Dirofilaria vectors such as Cx. pipiens s.l. are known to overwinter as mated females, which may lead to the quick development of Dirofilaria larvae with subsequent warming periods [42,44]. Hence, in order to prevent  Dirofilaria transmission in such areas, these factors have to be carefully considered when adopting chemoprophylaxis protocols, as already demonstrated in the field against Dirofilaria spp. infection in dogs [31].
The highest infection rate with D. immitis and D. repens was detected in Cx. pipiens s.l. mosquitoes (MIR = 0.6%, EIR = 0.6%) followed by Oc. caspius (MIR and EIR ranged from 0.1 to 0.4%), as already documented in central European Russia, Germany, Italy, Turkey and the Republic of Belarus [53]. High positivity of Cx. pipiens s.l. for D. immitis and D. repens was demonstrated by both molecular and parasitological studies [54,55]. Finally, the absence of Dirofilaria spp. DNA from Ae. vexans in the present study might be related to the smaller number of specimens examined (n = 6).

Conclusions
The present study highlights the sympatric occurrence of D. immitis and D. repens as well as the epidemiological pressure exerted by the length of the transmission season and the diversity of Dirofilaria spp. vectors in Corsica. We highlight public health risks, as Corsica attracts more than 750,000 visitors and their pets each year, which could pose an important risk for the transmission and spread of these zoonotic mosquito-borne filarioids. Moreover, the development of specific assays able to identify the infested/ infective mosquito species with Dirofilaria spp. are needed for an integrative surveillance approach.